An improved bimolecular fluorescence complementation assay with a high signal-to-noise ratio
An improved bimolecular fluorescence complementation assay with a high signal-to-noise ratio
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DOI：
10.2144/000113519
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发表时间：
2010-11-01
期刊：
影响因子：
2.7
通讯作者：
Hu, Chang-Deng
中科院分区：
文献类型：
--
作者：
Kodama, Yutaka;Hu, Chang-Deng
蛋白-蛋白相互作用（PPIs）在多种生物过程中起着至关重要的作用。在用于PPIs研究的生化、遗传和成像方法中，活细胞中PPIs的可视化是了解其细胞功能的关键。双分子荧光互补（BiFC）检测是直接可视化活细胞中PPIs的成像工具之一。BiFC分析是基于两个非荧光蛋白的N端和c端片段的结构互补，当它们融合到一对相互作用的蛋白上。尽管已有超过10种不同的荧光蛋白被用于BiFC检测，但所有这些荧光蛋白的两个非荧光片段都可以自发自组装，这有助于背景荧光，降低了BiFC检测中的信噪比。在这里，我们报告了一种突变I152L的鉴定，该突变可以特异性地减少自组装并降低基于金星的bic系统中的背景荧光。这种突变使得活细胞中bbifc测定的S/N比增加了4倍。这种改进的基于venus的bic系统将促进各种生物学研究领域的PPI研究。
Protein-protein interactions (PPIs) play crucial roles in various biological processes. Among biochemical, genetic, and imaging approaches that have been used for the study of PPIs, visualization of PPIs in living cells is the key to understanding their cellular functions. The bimolecular fluorescence complementation (BiFC) assay represents one of these imaging tools for direct visualization of PPIs in living cells. The BiFC assay is based on the structural complementation of two nonfluorescent N- and C-terminal fragments of a fluorescent protein when they are fused to a pair of interacting proteins. Although over 10 different fluorescent proteins have been used for BiFC assays, the two nonfluorescent fragments from all of these fluorescent proteins can spontaneously self-assemble, which contributes to background fluorescence and decreases the signal-to-noise (S/N) ratio in the BiFC assay. Here we report the identification of a mutation, I152L, that can specifically reduce self-assembly and decrease background fluorescence in a Venus-based BiFC system. This mutation allows a 4-fold increase in the S/N ratio of the BiFC assay in living cells. This improved Venus-based BiFC system will facilitate PPI studies in various biological research fields.